In article <2if1jb$80v at darkstar.UCSC.EDU>, smakk at cats.ucsc.edu (John Patrick
Latino) writes:
> An individual colony of E. coli 146 (deletion of gpt-lac), carrying a plasmid
> with genotype lac I-, Z-, pro+, was streaked onto a minimal lactose plate
> (lactose as sole carbon source). Presumably, it should not have formed any
> colonies, but in my lab group's case, it did (after incubation @ 37 deg.C for
> 48 hours. My question is: Since our strain was incapable of utilizing lactose
> because of a chromosomal deletion _and_ an amber base pair substitution in the
> episome, could the growth on lactose be due to a selection for revertants in
> the amber mutation?
>> We also plated colonies of the same strain on different
> media, including minimal glucose+X-Gal (which turns blue in the presence of
> B-galactosidase). In those cases, *white* colonies were observed. I would think
> that if the colonies on the lactose plates were revertants, then revertants
> would _also_ occur on the X-Gal plates, revealing blue colonies.
At a guess (it's been a while since I did this kind of lab work :-), you are
comparing streaking many bacteria (maybe 1x10^8 or 1x10^9) and seeing the
occasional revertant (back to wild-type is rare, I assume you have amber stops
in both lacI and lacZ on the plasmid, so they will not both revert, but amber
suppressor is of course possible). As your original colony is unable to grow on
this medium you can put a large number of bacteria on to select for mutants.
This way you see a very few rare mutants in a very large population.
In the second case, the starting strain *can* grow on the medium. You are
probably adding around 100 bacteria to the plate and spreading them so each
will grow as a single colony. To see a 1/10^9 event, you would therefore
need 10 million plates.
There are more complex possibilities of course. For example, would catabolite
repression, which lac is subject to, prevent the colour reaction with glucose
in the medium (streaking wild type E.coli will tell you the answer).
Biochemistry book never were much help with these cases (I was a biochemist
originally :-) Genetics textbooks are better , but only if you can follow them.
-----------------------------------------------------------------------------
Peter Rice, EMBL | Post: Computer Group
| European Molecular
Internet: Peter.Rice at EMBL-Heidelberg.DE | Biology Laboratory
| Postfach 10-2209
Phone: +49-6221-387247 | 69012 Heidelberg
Fax: +49-6221-387306 | Germany